Granulocyte Suspensions Research Articles

Heterogeneity in Chronic Granulomatous Disease Detected With an Improved Nitroblue Tetrazolium Slide Test

The microscopic nitroblue tetrazolium (NBT) slide test, used to score the ability of individual phagocytic leukocytes to produce superoxide, was improved according to the following procedure. Purified granulocyte suspensions are incubated with NBT and fixed in suspension, thereafter centrifuged on microscope slides and stained with nuclear fast red. This method precludes stimulation and selection of cells by adherence and washing. The number of formazan grains per cell can be judged in a semiquantitative way. In parallel incubations, the cells are stained with May-Grünwald/Giemsa, allowing identification of formazan-positive and -negative cells. The test discriminates well between cells from normal individuals, cells from patients with chronic granulomatous disease (CGD), and cells from heterozygotes for the X-linked form of CGD. Several patients and heterozygotes with an autosomal or variant form of CGD were detected with decreased NBT-reducing activity in their neutrophils and/or eosinophils. The relation between NBT-reductase activity of the phagocytes and the clinical situation is discussed.

Enhancement of Leukocyte Membrane Receptor Expression after Mechanical Agitation

Granulocyte and lymphocyte suspensions were exposed to slight mechanical agitation for 30 s on a tube mixer before being examined for membrane receptors by rosette techniques. Agitation caused an increase in the proportion of granulocytes bearing receptors for sheep erythrocytes (E-R), for the Fc portion of IgG (Fc gamma-R), and for C3b (C3b-R), and of lymphocytes bearing the Fc gamma-R. The proportion of E-R-bearing lymphocytes was not enhanced, with the exception of the 'early' or 'active' E-R-bearing cells. The increase in rosette formation was specific, i.e. rosette formation with ox or rabbit E was not induced by agitation. The increased proportion of membrane receptors was not associated with an increase in granulocyte phagocytosis of preopsonized zymosan particles, as measured by chemiluminescence. Membrane perturbation thus probably increases the availability of surface receptors.

Airways reversibility after prednisolone therapy in chronic asthma is associated with alterations in leukocyte function.

In 6 patients with chronic asthma, we studied the effects of orally administered prednisolone on monocyte and neutrophil membrane receptor expression for IgG (Fc) and complement, neutrophil locomotion, and calcium ionophore-induced leukotriene C4 production by granulocytes. Prior to treatment, monocyte IgG (Fc) and complement receptor expression, but not neutrophil IgG (Fc) or complement receptor expression, were increased when patients were compared with nonasthmatic control subjects (p less than 0.01 and p less than 0.01, respectively). After a 7-day course of orally administered prednisolone (30 mg/day daily), the increases in peak expiratory flow rate were accompanied by significant decreases from baseline in the percent of monocyte IgG (Fc) (Day 3, p less than 0.001; Day 7, p less than 0.001), monocyte complement (Day 3, p less than 0.001; Day 7, p less than 0.001), and neutrophil complement rosettes (Day 3, p less than 0.02; Day 7, p less than 0.02), but not neutrophil IgG (Fc) rosettes. Neutrophil locomotion in vitro, in response to casein, from the asthmatics was enhanced when compared with cells from control subjects, and was significantly depressed following prednisolone. Each of the 6 subjects had a peripheral blood eosinopenia in response to prednisolone that corresponded to a decrease in the amount of immunoreactive LTC4 generated from granulocyte suspensions challenged with the calcium ionophore A23187. These findings suggest that prednisolone suppresses monocyte and neutrophil activation in chronic asthma and that this is accompanied by an improvement in lung function.

Regulation of the oxidative response of human granulocytes to chemoattractants. No evidence for stimulated traffic of redox enzymes between endo and plasma membranes.

Experiments were performed to probe the role of exocytotic and endocytotic processes in the regulation of the human granulocyte O-2-generating system. Analytical subcellular fractionation studies indicated that 25-30% of the total cellular b-cytochrome and 8-10% of the flavin co-sedimented with plasma membrane markers, irrespective of stimulation of the cells by the chemoattractants N-formyl-Met-Leu-Phe (FMLP) or C5a. Phorbol myristate acetate stimulation resulted in significant translocation of b-cytochrome but not flavin from the specific granule/Golgi to the plasma membrane-enriched fractions. These results indicated that approximately 3.1 X 10(5) flavin and 0.8-1 X 10(6) b-cytochrome molecules are present in the plasma membrane of an isolated unstimulated human granulocyte and that these levels are invariant upon stimulation with chemoattractants. Maximal instantaneous rates of O-2 generation by cells in these preparations, however, were equivalent for all the stimuli. Since stimulation of granulocytes by phorbol myristate acetate, FMLP, or C5a results in exocytosis and/or endocytosis, then the role of these processes in regulating stimulated O-2 production by controlling the content of plasma membrane redox enzymes is questionable. This conclusion was supported by observations made with cytoplasts, which do not have an intracellular reserve of granules. Cytoplasts prepared from granulocytes produced O-2 at equivalent rates as their parent cells on a per unit surface area basis. These results suggest: 1) that stimulation of granulocytes with chemotactic peptides leads to full generation of O-2 at the cell surface without exocytotic recruitment of additional b-cytochrome and flavoprotein from the cytoplasmic compartment; 2) that these redox enzymes are not internalized along with chemoattractant receptors; and 3) that traffic of these redox enzymes between endo- and plasma membranes is not involved in the regulation of O-2 production in suspensions of human granulocytes stimulated by chemoattractants.

Intracellular freezing of human granulocytes

Human granulocyte suspensions were exposed to controlled freezing regimens on a cryomicroscope, and the incidence of intracellular freezing was measured as a function of cooling rate and extracellular nucleation temperature. The presence of intracellular ice was assessed by analysis of serially recorded images of the freeze-thaw process and by correlation with measured patterns of change in the cell volume. For granulocytes suspended in autologous plasma, a threshold was described for intracellular freezing as an empirical function of cooling rate ( B) and extracellular nucleation temperature ( T n): B (°C/min) = 1.1 T n (°C) + 12.3.

The effect of platelets and red cells on granulocytes stored at 22 degrees C.

Granulocyte concentrates contain varying numbers of platelets and red cells depending upon the method of collection. Either platelet or red cell concentrations may be as high as 2.0 X 10(12) per I. Studies were done on unwashed and washed granulocyte concentrates and on pure granulocyte suspensions with known numbers of platelets or red cells added. These suspensions or concentrates were stored for 72 hours at 22 degrees C. In both experiments, the following were measured: leukocyte and absolute granulocyte counts, dye exclusion, chemotaxis, plasma glucose, plasma pH, and osmolality. Red cell contamination did not adversely effect granulocyte storage. Platelets, however, did contribute to the functional deterioration of stored granulocytes. In the presence of high concentrations of platelets, both granulocyte dye exclusion and chemotaxis were adversely affected at 48 hours of storage. In another experiment, fresh autologous granulocytes incubated for 18 hours in hydroxyethyl starch-citrated-plasma obtained from stored granulocyte concentrates showed a progressive decrease in chemotaxis related to the age of the stored plasma. Glucose supplementation of the spent plasma maintained chemotactic activity. Contamination of granulocyte concentrates with other cells and the availability of glucose to granulocytes are variables affecting the short-term liquid storage of granulocytes at 22 degrees C.

In vitro release of lysozyme from monocytes and granulocytes.

When exposed to zymosan or latex particles or heat-inactivated staphylococci, freshly prepared human blood monocytes and granulocytes rapidly released a large fraction of their lysozyme content. Within 24 hours the total lysozyme activity in the monocyte suspensions tripled, while it doubled in the granulocyte suspensions, indicating synthesis of the enzyme following release. The monocytes in particular seemed to release and synthesize lysozyme without any other stimulus than contact with lymphocytes and the tube walls. Potassium caseinate in solution did not influence the lysozyme release. Myeloperoxidase and beta-glucuronidase, which in the granulocytes are kept in lysosomal fractions separate from most of the lysozyme, were neither released nor synthesized to a significant degree. Moreover, the minute amount of lactate dehydrogenase released indicated that the lysozyme release was not the result of cell lysis. Accordingly, the monocytes, which are not already stimulated by adherence to nonphagocytosable surfaces, are capable of selective enzyme release similar to that of the granulocytes.

A simple reliable assay for myeloperoxidase activity in mixed neutrophil-eosinophil cell suspensions: Application to detection of myeloperoxidase deficiency

Quantitation of myeloperoxidase (MPO) activity by guaiacol peroxidation (GP) assay is profoundly affected by the peroxidase present in eosinophils (EPO) that contaminate the granulocyte suspensions. Inclusion of 3-amino-1,2,4-triazole (AMT) in the GP assay permits quantitation of MPO activity in mixed neutrophil-eosinophil suspensions because of the differential inhibition of EPO and MPO by AMT. Results show that: (1) the peroxidase activity of eosinophil-free granulocyte suspensions is not appreciably affected by AMT; (2) in the presence of AMT the peroxidase activities of granulocyte preparations containing different numbers of eosinophils are similar on a neutrophil basis, regardless of the number of eosinophils and correspond with the activity of eosinophil-free granulocyte suspensions; (3) AMT almost completely inhibits the activity of partially purified EPO, only slightly affecting the catalytic activity of partially purified MPO; (4) AMT completely inhibits the residual peroxidase activity of granulocyte suspensions from MPO-deficient subjects contributed by contaminating eosinophils. The GP assay in the presence of AMT was used to study the pattern of hereditary transmission of MPO deficiency. The geneology derived on the basis of this assay was compatible with an autosomal recessive inheritance, in agreement with previously reported results, while no definite pattern of inheritance could be established by use of the GP assay without AMT. We suggest that the GP assay supplemented with AMT is the method of choice for detection of MPO deficiency, particularly partial deficiency.

Enzyme activity of isolated leukocyte populations. I. Cytochemical and zymographic studies of stored blood under various storage conditions

Heparinized venous blood was stored under sterile conditions at different temperatures (4 C, 20 C, 37 C) for various intervals (0-7 days). After storage the granulocytes and lymphocytes were isolated with routine methods. Naphthol AS-D-chloroacetate esterase as a granulocyte marker and acid alpha-naphthyl acetate esterase as a T-lymphocyte marker were identified on smears of the washed cell suspension. Different enzymes were identified in the cell sediment with electrophoresis. Relatively pure lymphocyte suspensions were obtained within the first 24 h. After this time, however, the percentage of these mononuclear cells declined markedly. The percentage of isolated granulocytes varied slightly; there was a marked predominance of granulocytes (more than 70%) at all intervals investigated during the isolation. Cytochemical analysis of the granulocytes and lymphocytes indicated that the decrease in the percentage of enzyme-positive cells depends in each case on the duration of the storage interval. During the first 24 h, only PGM1 and GOTM could be identified in the lymphocyte suspension with horizontal starch gel electrophoresis. The enzymes PGM1, PGM3, PGI, MDH, GOTM, 6-PGD, ADA could always be identified in the granulocyte suspension; AK, FUCA, MEM could be occasionally identified; and GPT and GLO could never be identified.

Enhancement of Chemotactic Migration by the Local Anesthetic Tetracaine

The local amine anesthetic tetracaine added to a suspension of guinea pig or human neutrophilic granulocytes inhibited their random migration in Boyden chambers, but increased their chemotactic migration towards the chemotactic tripeptide f-Met-Leu-Phe, complement-activated normal guinea pig serum, and the eosinophil chemotactic factor ECF. Tetracaine not only increased the distance migrated by the leading cells, it also caused more cells to leave the upper filter surface and to migrate into the filter. The effect required the presence of the drug; cells preincubated with tetracaine and washed did not differ from control cells. It is suggested that tetracaine specifically enhanced a mechanism operative in a cell's response to a concentration gradient of a chemotactic factor.

Mechanism of dexamethasone inhibition of chemotactic factor induced granulocyte aggregation

The reaction of FMLP with granulocytes causes aggregation and degranulation and enhances adherence to endothelium. To evaluate whether prevention of granule extrusion could impair these granulocyte activities, granulocytes were treated with either dexamethasone or hydrocortisone prior to treatment with FMLP. Dexamethasone was added to suspensions of cytochalasin B-treated granulocytes; it markedly impaired the aggregation response of the granulocytes of FMLP. When cytochalasin-B was not used, granulocyte aggregation in response to FMLP or PMA was inhibited by dexamethasone. Although dexamethasone prevented aggregation of cells following stimulation with FMLP or PMA, it failed to prevent the aggregation of granulocytes induced by rabbit lactoferrin. Adherence of granulocytes to human endothelial monolayers was enhanced by FMLP; dexamethasone inhibited the enhancement. However, with the addition of human lactoferrin to the granulocytes exposed to dexamethasone, the cells were able to adhere as well to endothelium as the cells exposed to FMLP but free of dexamethasone. When cytochalasin- B-treated granulocytes were incubated with dexamethasone or hydrocortisone prior to the addition of FMLP, the subsequent release of lactoferrin was substantially blocked, whereas the release of the primary granule products, lysozyme and beta-glucuronidase, was attenuated but not completely blocked. Thus, corticosteroids might block chemotactic-factor-induced granulocyte aggregation by selectively preventing release of specific granule products that contribute to and sustain aggregation.

Mechanism of dexamethasone inhibition of chemotactic factor induced granulocyte aggregation

The reaction of FMLP with granulocytes causes aggregation and degranulation and enhances adherence to endothelium. To evaluate whether prevention of granule extrusion could impair these granulocyte activities, granulocytes were treated with either dexamethasone or hydrocortisone prior to treatment with FMLP. Dexamethasone was added to suspensions of cytochalasin B-treated granulocytes; it markedly impaired the aggregation response of the granulocytes of FMLP. When cytochalasin-B was not used, granulocyte aggregation in response to FMLP or PMA was inhibited by dexamethasone. Although dexamethasone prevented aggregation of cells following stimulation with FMLP or PMA, it failed to prevent the aggregation of granulocytes induced by rabbit lactoferrin. Adherence of granulocytes to human endothelial monolayers was enhanced by FMLP; dexamethasone inhibited the enhancement. However, with the addition of human lactoferrin to the granulocytes exposed to dexamethasone, the cells were able to adhere as well to endothelium as the cells exposed to FMLP but free of dexamethasone. When cytochalasin-B-treated granulocytes were incubated with dexamethasone or hydrocortisone prior to the addition of FMLP, the subsequent release of lactoferrin was substantially blocked, whereas the release of the primary granule products, lysozyme and beta-glucuronidase, was attenuated but not completely blocked. Thus, corticosteroids might block chemotactic-factor-induced granulocyte aggregation by selectively preventing release of specific granule products that contribute to and sustain aggregation.

Stability of solutions of human granulocyte lactate dehydrogenase stored at different temperatures.

Granulocyte suspensions from eight healthy individuals were lysed ultrasonically in 0.15 M NaCl or serum. The leukolysates were tested for lactate dehydrogenase after being stored at 22 C, 5 C, -20 C and -196 C for one, two, three, four and seven days. The LDH content of granulocytes was 291 IU/10(10) cells (mean) and the enzyme had a characteristic isoenzyme pattern rich in LDH-5 with stepwise increases in each fraction from LDH-1 to LDH-5. The enzyme was unstable in saline solutions. This was marked at -20 C and related to the duration of storage. All the isoenzyme fractions showed significant inactivation. A mean 10% loss of activity occurred at -196 C. This loss was independent of the duration of storage and related to the process of freezing and thawing. Moderate inactivation occurred at room temperature while refrigerated samples were stable for four days. Serum protected the enzyme from denaturation and samples stored at -20, -196 and 22 C were stable for the seven days of the experiment.

Effects of temperature on granulocyte preservation

With the increasing use of granulocyte transfusion it is becoming important to determine if granulocytes can be preserved for a few days. If so, the optimum storage conditions must be identified. We studied the function in vitro of granulocytes collected as they would be for transfusion by continuous-flow centrifuge leukapheresis (CFCL) and filtration leukapheresis (FL). Granulocytes collected by CFCL maintained normal ability to phagocytose and kill bacteria after 48 hr and normal chemotaxis after 24 hr of storage at 20 degrees--24 degrees C. Neither 1 degrees--6 degrees C nor 37 degrees C were as effective in preserving chemotactic response. Agitation of the granulocyte suspension during storage caused reduced bacterial killing and chemotaxis. Granulocytes collected by FL functioned very poorly after 24 hr storage at all temperatures studied. These studies suggest that it may be possible to store CFCL granulocytes at 20 degrees--24 degrees C for 24 hr. FL granulocytes should not be stored at all.

Effects of temperature on granulocyte preservation

Abstract With the increasing use of granulocyte transfusion it is becoming important to determine if granulocytes can be preserved for a few days. If so, the optimum storage conditions must be identified. We studied the function in vitro of granulocytes collected as they would be for transfusion by continuous-flow centrifuge leukapheresis (CFCL) and filtration leukapheresis (FL). Granulocytes collected by CFCL maintained normal ability to phagocytose and kill bacteria after 48 hr and normal chemotaxis after 24 hr of storage at 20 degrees--24 degrees C. Neither 1 degrees--6 degrees C nor 37 degrees C were as effective in preserving chemotactic response. Agitation of the granulocyte suspension during storage caused reduced bacterial killing and chemotaxis. Granulocytes collected by FL functioned very poorly after 24 hr storage at all temperatures studied. These studies suggest that it may be possible to store CFCL granulocytes at 20 degrees--24 degrees C for 24 hr. FL granulocytes should not be stored at all.

Large scale preparation of leukocyte concentrates and further purification of lymphocytes and granulocytes with the IBM 2991 cell processor.

A procedure is described for the large-scale production of pure suspensions of human lymphocytes and granulocytes. Platelet-poor buffy-coats, obtained by centrifugation of six units of ABO-compatible ACD blood, were pooled and centrifuged again, resulting in a crude leukocyte concentrate containing approximately 60 per cent of the original amount of leukocytes. Further fractionation of such crude leukocyte concentrates was achieved by scaling up Bøyum's Ficoll-Isopaque density gradient technique with the aid of an IBM 2991 blood cell processor. In this way 2.9 +/- 0.3 X 10(9) mononuclear leukocytes, consisting of 90 +/- 0.7 per cent lymphocytes and 5.3 +/- 0.3 X 10(9) polymorphonuclear leukocytes of 92 +/- 0.7 per cent purity could be isolated in one run. The overall yield was 36 per cent of the original amount of leukocytes.

The isolation of ovine lymphocytes and granulocytes from whole blood using hydroxyethylcellulose

Sheep erythrocytes do not aggregate spontaneously but will sediment when mixed with a solution of hydroxyethylcellulose. The resulting leucocyte-rich supernatant can be used in the preparation of purified lymphocyte and granulocyte suspensions.

Inhibition by 5-hydroxytryptamine of anaphylactic histamine release from bovine granulocytes

A suspension of granulocytes from calves sensitised to horse plasma released histamine when exposed to the antigen. Histamine release was strongly inhibited by 5-hydroxytryptamine in a dose-related fashion. 5-HT is known to be released from bovine lungs in vitro, and may regulate the release of histamine in vivo during anaphylaxis.

Leukocyte-platelet interaction. Release of hydrogen peroxide by granulocytes as a modulator of platelet reactions.

Because of the many potent biological capabilities of the blood granulocytes, and their contact with platelets in various physiologic and pathologic states, a possible interaction between granulocytes and platelets was investigated. Platelets were purified by gel filtration and via a dialysis membrane were separated from suspensions of autologous granulocytes prepared by dextran sedimentation and resuspended in modified Tyrode's buffer. After 20 min at 37 degrees C platelet aggregation was shown to be diminished by such exposure, as compared to the aggregation of platelets incubated with dialysates of buffer only. When granulocytes were stimulated by the addition of 1.1-muM latex spheres as target particles for phagocytes, the dialysate of these cells exhibited greatly enhanced platelet-inhibitory properties. The addition of catalase to the platelets abolished the effect of exposing these cells to the dialysate of resting granulocytes and markedly inhibited the effect of exposing the platelets to the dialysate of phagocytosing granulocytes. Catalase treated with 3-amino-1,2,4-triazole had no platelet-protective capacity. Purified suspensions of lymphocytes released no platelet-inhibitory principle under these experimental conditions. Hydrogen peroxide in the dialysate of granulocytes was measured directly with an assay involving an H2O2-induced decrease in the fluorescence of scopoletin catalyzed by horseradish peroxidase. The dialysate of phagocytosing granulocytes contained 0.86 +/- 0.55 nmol H2O2/2.5 X 10(7) granulocytes when sampled at 20 min. By an alternate measurement technique in which scopoletin and horseradish peroxidase were present in the dialysate from time zero, the mean amount of H2O2 in the dialysate reached 4.0 +/- 1.3 nmol/2.5 x 10(7) granulocytes at 20 min. This discrepancy suggested the consumption of H2O2, possibly mediated by the granulocytes themselves. This possibility was investigated by the addition of exogenous H2O2 to the test system. Both granulocytes and platelets enhanced the disappearance of H2O2 from the dialysate, and the amount consumed was proportional to the amount of H2O2 added to the system. Glucose oxidase at 12 M U/ml plus glucose in excess resulted in the production of H2O2 at a rate and final amount comparable to that produced by phagocytosing granulocytes. This mixture, when substituted for phagocytosing granulocytes in the standard dialysis membrane experiment, induced an inhibition of platelet aggregation similar to that caused by the granulocytes. The observation that the release of H2O2 by the blood granulocyte influences platelet function suggests a potential role for the granulocyte in the regulation of hemostasis or thrombosis.

Antibody-dependent human lymphocytotoxicity: a micro assay system.

A micro assay system is described for measuring antibody-dependent lymphocytotoxicity with human effector cells. The target cells were mouse mastocytoma cells sensitized with rabbit antibody. The tests were performed in microtiter plates, each well containing 5 x 10(3) 51Cr-labeled target cells, together with varying numbers of effector cells. Whereas highly purified lymphocyte suspensions induced lysis, purified monocyte and granulocyte suspensions had virtually no effect. In parallel experiments it was shown that the latter cell suspensions were very active in lysing sensitized human erythrocytes, which in turn were not lysed by purified lymphocytes. Finally, it was shown that the lytic capacity of lymphocytes was not affected by preservation of the cells at liquid nitrogen temperature. It is concluded that the system described can be used for the clinical analysis of antibody-dependent lymphocytotoxicity in lymphocyte suspensions.